AATP was synthesized through a substitution reaction of acryloyl chloride. Meanwhile, poly (PTAm) was prepared as a control sample. Then, the structures of products were characterized by nuclear magnetic resonance spectroscopy (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), high performance liquid chromatography-mass spectrometry (HPLC-MS), differential scanning calorimetry (DSC), X-Ray photoelectron spectroscopy (XPS), and electron paramagnetic resonance (EPR), respectively. Additionally, the electrochemical impedance spectra (EIS) and the charge-discharge cycling properties were studied. The results demonstrated that the poly(TAm-co-SSS) with the side group of sodium sulfonate adjacent to TEMPO group exhibits a better charge-discharge cycling stability than that of the PTAm. Moreover, the charge specific capacity of the poly(TAm-co-SSS) is larger than that of the PTAm. Besides, the first coulombic efficiency of poly(TAm-co-SSS) is higher in comparison with that of PTAm. These superior electrochemical performances were ascribed to the synergistic effect of sulfonate ions group and nitroxyl radical structure, which benefits the improvement of charge carrier transportation of the nitroxyl radical polymers. Consequently, the nitroxyl radical poly(TAm-co-SSS) is promising for use in organic radical battery materials, based on the good electrochemical properties.A novel nitroxyl radical polymer poly(TEMPO-acrylamide- Organic-based electrode-materials can ensure the safety and cycle performance of a lithium secondary battery, and have the characteristics of bendable deformation, high specific energy, wide operating temperature range, long storage life, small self-discharge, and no memory effect, and can be used according to requirements ,2,3,4,5.As a typical radical polymer, poly (PTAm) indicates that it can be used as a novel, metal-free cathode material or long-period rechargeable devices ,14. Moreco-SSS). This would allow the polymer material to adapt to a wider range of electrolyte solutions and different application requirements in electronic devices. The structure and electrical properties of poly(TAm-co-SSS) were investigated. The charge-discharge properties of the prepared polymers were studied by galvanostatic charge-discharge testing. The radical polymer poly(TAm-co-SSS) exhibits good electrochemical lithium storage performance. We demonstrated that the novel radical polymer poly(TAm-co-SSS) can be used in an organic radical battery. These results highlight the ability to expand the molecular doping paradigm beyond the conjugated polymer regime and into the realm of radical polymers, such that materials can be synthesized in a ready and systematic fashion. It is vital to scale up production of the organic radical polymer materials.We extended the paradigm to the realm of radical polymers through the intentional addition of ion-bearing repeat units to the macromolecular architecture of PTAm. Then, these ion-bearing repeat units were introduced in order to enhance the charge-discharge cycling stability of the copolymers relative to pristine PTAm. This objective of this work was to modify PTAm with sodium styrene sulfonate—a reactive group—to obtain a hydrophilic polymer radical polymer poly, tetrahydrofuran and the remaining reagents that were of analytical grade were bought from Shanghai Macklin Biochemical Co., Ltd. .4-Amino-2,2,6,6-tetramethylpiperidine , toluene , acryloyl chloride , azobisisobutyronitrile (AIBN), and styrene sulfonate  were bought from Aladdin Industrial Co., Shanghai, China; co-SSS)) and PTAm is shown in The synthesis route of the polymer  was poured into the toluene while magnetic stirring was working. After stirring for more than 10 min, acryloyl chloride  was slowly added dropwise to the flask within 30 min. Then the mixture was stirred in the ice bath for 1 h, and then at room temperature for 1 h. When the reaction was over, the resulting turbid liquid was filtered with a vacuum pump to collect a white precipitate. The precipitate was washed three times with dichloromethane, and then washed with a saturated NaHCO3 solution. The dichloromethane was removed by vacuum drying for 24 h. The final product was recrystallized from methanol after insoluble impurities were removed, as was water by addition of anhydrous MgSO4 for more than 1 h and filtration; the final product was a white crystal [The molecular sieve-treated anhydrous toluene (90 mL) was poured into a 100 mL round-bottomed flask, covered with a plastic stopper, and sealed with a tape. The flask was placed on a magnetic stirrer with an ice bath (T < 5 °C). The flask was pinched in the plastic plug and purged N crystal .n-hexane with stirring to remove AATP, SSS, and AIBN. A white precipitate was obtained after suction filtration; then, it was dried in a vacuum oven for 24 h to remove the residual n-hexane. A white powder poly(AATP-co-SSS) was finally obtained. In the same time, radical polymer PAATP was synthesized by referring to the literature [AATP  and SSS   = 1:1) were poured into a Schlenk tube containing methanol aqueous solution , AIBN  under magnetic stirring until the solid dissolved completely. The above mixed solution was frozen in liquid nitrogen. The Schlenk apparatus with a double-row tube was used to pump air through the test tube suction port. In the same time that nitrogen gas was introduced to the Schlenk tube, the frozen solution completely melted. Then, the above operation was repeated until no bubbles were generated in the solution. The test tube was purged with nitrogen, while it was placed in a 65 °C oil bath with stirring for 10 h. After that, the reaction product was poured into terature .co-SSS) (100 mg) was dispersed completely into 5 mL of tetrahydrofuran (THF) in a 100 mL three-necked flask equipped with a constant pressure separatory funnel under ultrasonic vibration. Then, mCPBA (200 mg) dissolved in THF (5 mL) was added into the flask through the funnel. Then, the mixed solution was stirred for 7 h in a nitrogen atmosphere. After that, the mixture was washed with 20% Na2CO3 solution. The separated organic phase solution was slowly dropped into a n-hexane solution to precipitate a product. Meantime, the radical polymer PTAm was synthesized according to the literature [The poly using deuterated chloroform. FT-IR spectra were carried out on a VECTOR-22 spectrometer  with KBr pellets at wavelength range from 500 cm−1 to 4000 cm−1. XPS measurements were taken using a AXIS X-ray photoelectron spectroscopy . Electron paramagnetic resonance (EPR) was carried out on an E500 Electron paramagnetic resonance  at a concentration of 2 mg·mL−1 in tetrahydrofuran (THF) at room temperature. DSC measurements were carried out on the as prepared samples, using a Discovery DSC 250 series instrument . The samples were initially heated to 200 °C and cooled to −50 °C at 10 °C·min−1 under a nitrogen gas purge. The data shown were from the final scans from −50 °C to 200 °C at 10 °C·min−1.The monomer AATP was characterized by high performance liquid chromatography-mass spectrometry (HPLC-MS) . co-SSS) and PTAm were, respectively, mixed with acetylene black and binder (PVDF) in a mass ratio (8:1:1). Then, a small amount of N-methyl pyrrolidone was added dropwise into the above mixtures with stirring to form a uniform paste. The paste was placed on an aluminum foil and dried under vacuum at 70 °C for 12 h. And then, the dried paste was cut into slices as positive electrodes. CR2025 coin-type batteries were assembled using a lithium metal sheet as the counter electrode and negative electrode, l mol·L−1 LiPF6/EC+DMC (1:1) solution as an electrolyte, and celgard 2400 as the diaphragm in an argon filled glove box. The charge and discharge of the battery were tested by the BTS-XWJ-6.44S-00052 multi-channel battery program control tester  at the voltage range from 2.0–4.3 V with the current density of 100 mA·g−1 at room temperature. The electrochemical impedance spectra of the battery were recorded on a CPARSTATMC electrochemical workstation  at an open circuit potential with the voltage amplitude of 10 mV and the frequency range from 100 kHz to 0.1 Hz.The prepared poly(TAm-1H-NMR spectrum of the prepared AATP is displayed in 2), 6.08 , 5.67 , 5.32 , 4.37 , 1.97 , 1.29 , 1.15 , 0.97 . According to the reference [The eference ,26, it w−1 was assigned to the secondary amine N–H stretching vibration peak; 1558.94 cm−1 corresponds to the amide NH bending vibration peak; 2953.75 cm−1, 2881.56 cm−1, and 2873.16 cm−1 are, respectively, ascribable to the stretching vibrational peaks of –CH3, –CH2, and –CH on the piperidine ring; 1653.50 cm−1 was attributed to the amide C=O stretching vibration peak; and 1618.23 cm−1 is related to the stretching vibration peak of the double bond of C=C.The FT-IR spectrum of the prepared AATP is shown in 1H-NMR, and HPLC-MS, it was confirmed that the product prepared was the AATP monomer [The HPLC-MS spectra of the monomer AATP are shown in  monomer .1H-NMR analyses were carried out to identity the copolymers poly(AATP-co-SSS) and PAATP. The characteristic spectra are displayed in co-SSS) and PAATP, the peak at 4.20 ppm was assigned to C–H (a) on piperidine attached to amide group. The broad peak at 1.08 ppm is ascribable to the chemical shifts of four methyl-side groups (b) of piperidine ring. However, the peak of 7.60 ppm which corresponds to hydrogen from the benzene ring (c) was only in the spectrum of poly(AATP-co-SSS). Besides, the C=C bonds of AATP and SSS were participating in the polymerization; they became single bonds. That indicates that AATP and SSS were successfully copolymerized to form the polymer poly(AATP-co-SSS).The co-SSS), and poly(TAm-co-SSS) were found as shown in −1 was assigned to an amide N–H bending vibration peak; 1650 cm−1 was attributed to an amide C=O stretching vibration peak; 1460 cm−1 was attributed to an N–O stretching vibration peak; 1180 cm−1 and 1040 cm−1 respectively, correspond to the antisymmetric and symmetric stretching vibration absorption peaks of sulfonate. Compared with the monomer AATP infrared spectrum (co-SSS) [co-SSS) spectrum, the absorption peak of the N–O stretching vibration peak became significantly stronger. It resulted from the secondary amine N–H bond in pyridine ring being oxidized to N–O radical. It also proved that the oxidation product poly(TAm-co-SSS) was obtained. The FT-IR characteristic peaks of PTAm, poly . Compareco-SSS) because the chemical bond N-H in pyridine of the polymer has participated in the oxidation reaction. By X-ray photoelectron spectroscopy analysis of poly(AATP-co-SSS) and poly(TAm-co-SSS), the 399.8 eV peak in the whole spectrum is assigned to the binding energy of N–H bond in N1s in the polymer [co-SSS) at this position, the significantly weak absorption intensity of poly(TAm-co-SSS) indicates that the N–H of pyridine ring in poly(AATP-co-SSS) is oxidized to N–O• in poly(TAm-co-SSS) [co-SSS) illustrates two different binding energy peaks including 399.8 eV and 401.0 eV correspond to a protected nitrogen (N–H or CO–NH) and a N–O• functional group, respectively. It is confirmed that the N-H of pyridine ring in poly(AATP-co-SSS) is oxidized to N–O• in poly(TAm-co-SSS). Moreover, according to the chemical structure of poly(AATP-co-SSS), N–H quantity of pyridine is equal to that of CO–NH. By quantification analysis of N–O and N–H bond of poly(TAm-co-SSS), N–H bond of pyridine has almost been oxidized completely because the area of N–O peak is basically equal to N-H peak from CO–NH.As we can see from  polymer . Compare-co-SSS) . Furtherco-SSS) and PTAm. co-SSS) and PTAm, respectively. The result shows the hyperfine coupling constant g values of poly(TAm-co-SSS) and PTAm are both 2.0047, which was calculated according to the formula hν = gβH corresponding to that of TEMPO (2.0056) [co-SSS). However, its spin-spin exchange and dipole–dipole interactions between molecules or molecules are relatively weakened, which may be due to the TEMPO moiety in the copolymer was partly replaced by sulfonate structure [co-SSS) can be clearly distinguished by the external SSS section. Furthermore, the EPR spectrum of the polymer poly(TAm-co-SSS) shows a little broader peak at H = 3505 than that of PTAm. The difference in line width of nitroxyl radicals between poly(TAm-co-SSS) and PTAm is mainly due to the constant transition of electrons between two energy levels, which is a dynamic equilibrium process [The nitroxides polymers are paramagnetic because of stable nitroxides. Thus, EPR was used to determine the N–O• radicals of poly(TAm-(2.0056) . This prtructure . By quan process .co-SSS) and PTAm are shown in DSC thermograms corresponding to poly(TAm-co-SSS) exhibits a distinct semicircle in the high frequency range. The radius of the semicircle indicates the impedance of the poly(TAm-co-SSS) electrode when it is electrochemically polarized, and presents a straight line at the low frequency band [co-SSS) is 223 Ω, which is almost similar to that of PTAm is 218 Ω. The possible reason is that the nitroxyl radical polymer containing ion holes is activated in the test processing [It is generally believed that the AC impedance spectrum is composed of a straight line located in the low frequency region and a semicircular curve located in the high frequency region. The body resistance Rb corresponds to the diameter of the semi-circular curve of the high frequency region of the system. Meanwhile, the smaller the diameter is, the smaller the body resistance of the corresponding system is. That is, the conductivity is the higher. ncy band . The semocessing . The dopocessing . This poco-SSS) at the current density of 100 mA·g−1. The test conditions of the poly(TAm-co-SSS) as a lithium positive electrode material were the same as those of the PTAm. Meantime, the poly(TAm-co-SSS) had charge and discharge curves similar to that of the PTAm as a comparative sample. At the initial stabilization cycle, the capacity was slightly attenuated. As the cycle progressed, the capacity of poly(TAm-co-SSS) increased to some extent. After 10 charging and discharging cycles, the capacity began to stabilize. That may be because the solid electrolyte interface (SEI) film which formed in the lithium battery hindered the cycle progress of charge and discharge at the first cycle. After that, the carbon structure on the backbone of the nitroxyl radical polymer was gradually activated to be a polymer structure with more electron transfer active sites. It is worth mentioning that the reversible specific capacity of poly(TAm-co-SSS) as an active electrode material is 95.7 mAh·g−1. After 100 cycles, the specific capacity of poly(TAm-co-SSS) was maintained at 95 mAh·g−1 (the retention is over 95%), which is higher than of the PTAm. That indicates that the poly(TAm-co-SSS) has a better charge-discharge cycling stability than that of the PTAm. Moreover, the first coulombic efficiency (78%) of poly(TAm-co-SSS) was higher in comparison with that of PTAm (52%). So the poly(TAm-co-SSS) has better electrochemical performance superior to the PTAm. It is may due to the synergistic effect of sulfonate ions group and nitroxyl radical structure which has a great influence on the nitroxyl radical polymer. The sulfonate ions group which was introduced into PTAm not only increases the electron mobility, but also facilitates the transport of lithium ions [+ hosting group to improve exchange of Li+ ions [co-SSS) exhibits good electrochemistry Li-ion storage performance.ium ions . PTAm coLi+ ions . MoreoveLi+ ions . Consequco-SSS) also verifies the stability performance of charge-discharge cycling. As can be seen in co-SSS) (b) obviously changes from that of PTAm. A medium-magnification  SEM image of poly(TAm-co-SSS) clearly shows that the sample has a looser stacking structure. The electrode material with a loose assemble particle and large special surface area is in favor of the utilization of electrode-active material as well as the improvement of the electrochemical properties [The morphologies of the as-obtained polymers and cathode material before and after the cycling test have also been investigated by SEM. The morphology changes from PTAm to poly was obtained by free radical polymerization with sodium styrene sulfonate (SSS) and AATP. After oxidation, a faint yellow solid poly(TAm-co-SSS) was formed and characterized by FT-IR and XPS. The structure confirms that the product synthesized was the target product. By performing a button cell assembly on the synthesized radical polymer poly(TAm-co-SSS), the capacity and electrochemical performance tests were carried out. Adjusting pendant groups and medium crossing structure can enhance the transport ability and improve the charge-discharge cycling stability. The TEMPO-substituted polyacrylamide bearing sulfonate sodium pendant is expected to be used as an organic electrode material. It provides a vital pathway by which to optimize next-generation radical polymer designs.In this work, a paradigm was extended to the realm of radical polymers through the intentional addition of ion-bearing repeat units to the macromolecular architecture of the radical polymer PTAm. As its key monomer, 4-acrylamido-2,2,6,6-tetramethylpiperidine was prepared using 4-amino-2,2,6,6-tetramethyl-piperidine and acryloyl chloride. The AATP was characterized by


Colorectal peritoneal metastases (CPM) occur in up to 13% of patients with colorectal cancer, presenting either synchronously or metachronously. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS and HIPEC) have been increasingly utilised for selected CPM patients with favourable outcomes, though its benefits may differ for synchronous (s-CPM) and metachronous CPM (m-CPM).A retrospective analysis of CPM patients treated with CRS and HIPEC at the National Cancer Centre Singapore over 15 years was performed. In the s-CPM group, CPM was diagnosed at primary presentation with CRS and HIPEC performed at the time of or within 6 months from primary surgery. In the m-CPM group, patients developed CPM > 6 months after primary curative surgery.p = 0.619). Median overall survival was significantly prolonged in patients with m-CPM . In a subset of m-CPM patients with limited PCI in whom ICU stay was not required, a survival advantage was seen (p = 0.031).One hundred two patients with CPM were treated with CRS and HIPEC. Twenty (19.6%) patients had s-CPM and 82 (80.4%) had m-CPM. Recurrences occurred in 45% of s-CPM and in 54% of m-CPM (A survival advantage was seen a subset of m-CPM patients, possibly representing differences in disease biology. Peritoneal metastases (PM) occur in up to 20% of patients with stage IV colorectal cancer (CRC) . The perIn CRC with hepatic metastases, it has been proposed that a synchronous or metachronous presentation implied differing tumour biologies . SynchroAs such, our study aims to compare survival and recurrence outcomes of patients with s-CPM and m-CPM in the context of CRS and HIPEC. We believe that discussing their outcomes independently may shed light on their possibly distinct biology and is crucial in any management algorithm undertaken.The current study was performed in a single tertiary institution. Data was retrieved from a prospectively maintained database of patients treated with CRS and HIPEC for CPM between January 2003 and January 2018.Our primary endpoints were overall survival (OS) and progression-free survival (PFS). Clinical characteristics, operative data, and 30-day morbidity and mortality were also evaluated.The study was conducted with the approval of the Centralized Institutional Review Board of Singapore Health Services.Synchronous CPM (s-CPM) – CPM present at the time of the first diagnosis for which CRS and HIPEC were performed within 6 months of diagnosisnot present at the time of the first diagnosis of colorectal cancer but detected at subsequent follow-up and for which CRS and HIPEC were performedMetachronous CPM (m-CPM) – CPM was Patients were classified into two groups for comparison:In both groups, OS was defined as time in months, between CRS and HIPEC to date of last follow-up or death, while PFS was defined as the time interval from the date of CRS and HIPEC to the date of detection of recurrent disease.The Peritoneal Cancer Index (PCI) score as described by Sugarbaker was used to describe the extent of disease . The comThe decision for upfront CRS and HIPEC versus neoadjuvant treatment prior to surgery was guided by a multi-disciplinary tumour board comprising of surgical, medical and radiation oncologists, pathologists and radiologists. Patient fitness and preference; tumour biology, inferred by PCI score; disease-free interval; primary tumour characteristics ; and confidence of surgeon to achieve a CC0 resection were all considerations in the selection of patients for pre-operative systemic therapy.2 for males and 10 mg/m2 for females) with 4 L of peritoneal dialysis solution at 41–42 °C over a duration of 60 min. A hyperthermia pump was used during the study duration to deliver the intraperitoneal chemotherapy agent via a single inflow catheter, and drainage was via four intra-abdominal drains.The CRS and HIPEC procedure performed at our institution was as previously described , 16 and Post-operatively, patients were transferred to the surgical intensive care unit (SICU) or high-dependency unit for monitoring. All intra- and post-operative complications were recorded and graded based on the Clavien-Dindo classification .During follow-up, patients were reviewed at 3 monthly intervals during which a full physical examination and tumour markers were taken. A computed tomography (CT) scan of the chest-abdomen and pelvis was performed 6 monthly for the first 2 years post CRS-HIPEC and then yearly thereafter. Details of recurrences, if any, were recorded.t tests for numeric variables. Wilcoxon rank-sum test was used if the distributions were skewed for numeric variables. Survival functions were estimated using Kaplan-Meier method, and log-rank test was used to evaluate the differences between the two groups. Univariate Cox regression was applied to investigate potential factors on the risk of the event of death and recurrence. Variables with p value less than 0.10 in the univariate analysis were included in the multivariate Cox regression model. The model was built using backward selection. Variables with p value < 0.05 would be in the final model. Cox proportionality assumption was assessed by using an overall test on Schoenfeld residuals.Differences in demographics and clinical characteristics of the patients and recurrent patients were assessed between the two groups (1) s-CPM and (2) m-CPM by using Fisher’s exact test for categorical variables and two group p value of < 0.05 was considered statistically significant. All analyses were performed using Stata version 12.0.A two-sided p = 0.043). Other baseline clinical-pathological characteristics are as described in Table A total of 102 patients with CPM underwent CRS and HIPEC from January 2003 to January 2018. There were 20 (20%) patients with s-CPM and 82 (80%) with m-CPM. Ninety-seven percent of all patients with CPM and all with s-CPM had locally advanced, i.e. T3/T4 primary tumours. Pre-operative CEA levels was significantly higher in the s-CPM patients (n = 15) received neo-adjuvant chemotherapy or chemoradiation therapy before CRS and HIPEC were performed. Agents used were mainly 5-fluorouracil (FU) based with the addition of oxaliplatin or irinotecan. Targeted agents were used at the discretion of the medical oncologists after consideration of general response and molecular status. In the m-CPM group, the median time between surgery for the primary tumour and the development of metachronous metastases was 21.7 months (range 7.9–186.2). Eleven (13%) patients received neoadjuvant chemotherapy prior to CRS and HIPEC.In the s-CPM group, 75% (p = 0.618). There were no differences in terms of intra-operative blood loss (1200 ml versus 1003 ml).The CRS and HIPEC duration was 390 min (range 245–855) in s-CPM and was 415 min (range 200–960) in m-CPM (p = 0.12). All patients received complete cytoreduction surgery.Overall, the median PCI score was 7 (range 0–27). There was no significant difference in the PCI scores when comparing s-CPM and m-CPM  patients received further systemic therapy, of which nine patients were from the s-CPM group and 19 the m-CPM group.p = 0.198). Post-operative complications occurred in 47% (n = 48) of patients with no difference between the two groups. Majority of patients (86%) suffered Clavein-Dindo grade 1 or 2 complications. Of the 15 patients who required invasive intervention (i.e. grade 3 or 4 complications), four patients suffered pleural effusions necessitating chest tube insertion; two patients had post-operative bleeding needing re-laparotomy; and two patients had a ureteric leak requiring insertion of percutaneous nephrostomy (PCN) tubes, with three patients having intra-abdominal collections, three patients with anastomotic leak requiring insertion of abdominal drain and one patient with acute retention of urine requiring the insertion of urinary catheter. There was no in-hospital mortality.Overall, the median duration of hospital stay was 12 days (range 7–66), and it was 14.5 days (range 7–26) and 11 days (range 7–66) in s-CPM and m-CPM patients, respectively  from the s-CPM group and 45  from the m-CPM group (p = 0.619). Median time to recurrence was 9.5 months (range 0.9–33.7). It was 13.1 (range 2.6–17.8) and 9.5 (range 0.9–33.7) months respectively in s-CPM and m-CPM groups (p = 0.917). The pattern of recurrence was as described in Table In total, 54 52.4%) patients developed recurrences after CRS and HIPEC: nine . One-, 3-, and 5-year OS is as illustrated in Table Overall median OS in all CPM patients was 40.6 months. Comparing s-CPM and m-CPM, a significant difference was found in OS outcomes: 26.9 months (range 9.8–44.5) in the former and 45.2 months range 30.2–54.3) in the latter . In addition, in the subset of m-CPM patients with PCI < 12, a more significant survival advantage was seen (Table On subgroup analysis adjusted for ICU stay, it was found that in patients who did not require ICU stay, s-CPM was associated with poorer OS (en Table .Table 5The tumour cell entrapment hypothesis has been postulated to be the mechanism behind the occurrence of peritoneal disease . LocallyBoth synchronous and metachronous PM are known indications for CRS and HIPEC in CRC. Unlike hepatic, pulmonary and para-aortic lymph node metastases for which synchronous disease have been found to be associated with an aggressive biology and poorer survival , 20, 21,At present, rates of s-CPM in published studies range from 20% to 80%. In the PRODIGE 7 trial , 30% andRecurrence despite CRS and HIPEC is common, occurring in up to 65% of patients with CPM . In our The retrospective design and relatively small numbers in this study may have resulted in selection bias as well as a failure to show a significant difference between s-CPM and m-CPM groups in the multi-variate analysis. Though subgroup analysis pointing to a possible trend improves OS in the m-CPM group after accounting for the PCI score and ICU stay, further prospective studies with larger sample sizes will be necessary to further elucidate the true biological differences between s-CPM and m-CPM.In a subset of m-CPM patients with limited peritoneal disease in whom intensive care post-operatively was not required, a survival advantage was seen over the s-CPM group. This may represent differences in disease biology and emphasises the need to approach these patients differently. Further prospective studies are needed to determine the appropriate management of s-CPM versus m-CPM.


The simultaneous multi-slice EPI  sequence has met immense popularity recently in functional neuroimaging. A still less common alternative is the use of 3D-EPI, which offers similar acceleration capabilities. The aim of this work was to compare the SMS-EPI and the 3D-EPI sequences in terms of sampling strategies for the detection of task-evoked activations at 7T using detection theory. To this end, the spatial and temporal resolutions of the sequences were matched  and their excitation profiles were homogenized by means of calibration-free parallel-transmission . We used a fast-event “localizer” paradigm of 5:20 min in order to probe sensorimotor functions  as well as higher level functions , where results from a previous large-scale study at 3T (N = 81) served as ground-truth reference for the brain areas implicated in each cognitive function. In the current study, ten subjects were scanned while their activation maps were generated for each cognitive function with the GLM analysis. The SMS-EPI and 3D-EPI sequences were compared in terms of raw tSNR, t-score testing for the mean signal, activation strength and accuracy of the robust sensorimotor functions. To this end, the sensitivity and specificity of these contrasts were computed by comparing their activation maps to the reference brain areas obtained in the 3T study. Estimated flip angle distributions in the brain reported a normalized root mean square deviation from the target value below 10% for both sequences. The analysis of the t-score testing for the mean signal revealed temporal noise correlations, suggesting the use of this metric instead of the traditional tSNR for testing fMRI sequences. The SMS-EPI and 3D-EPI thereby yielded similar performance from a detection theory perspective. With ultra-high fields (UHF), functional MRI (fMRI) benefits from increased signal-to-noise ratio (SNR) and sensitivity to BOLD contrast so that higher spatial resolutions can be reached. However, to maintain high temporal sampling rates for high spatial resolution, whole-brain coverage, the use of fast k-space sampling strategies becomes mandatory. Simultaneous multi-slice EPI (SMS-EPI) , 2 has m+ field inhomogeneity. This can result in local signal voids, impairing signal and hence tSNR most dominantly in the temporal lobes and cerebellum. In addition, the specific flip angle excitation patterns of the SMS-EPI and 3D-EPI sequences result in different signal responses that play a role in the comparison of these sequences, especially at 7T. Several approaches have been proposed to achieve homogeneous excitation flip angles across the whole volumes, such as dielectric pads [+ mapping, static field (ΔB0) mapping, brain masking and on-the-fly RF pulse design to calculate the optimal RF and gradient waveforms. The concept of universal pulses (UP) was recently introduced in order to skip this time-consuming step and simplify the acquisition workflow [+ and ΔB0 over subjects: using a database of B1+ and ΔB0 maps from a collection of subjects, the pulses are designed to achieve the best possible flip angle homogeneity over this representative population for given hardware and SAR constraints. The resulting UPs can then easily be applied on any subject without the need for subject-specific calibration scan or pTx-related operator interactions. Following previous demonstrations of UPs in anatomical imaging sequences  and their robustness across multiple sites [Despite the dominating benefit of superior tSNR and BOLD sensitivity at UHF, a considerable impediment is the transmit B1ric pads  and pararic pads , 19. In ric pads , 21 or nric pads . For a rric pads . The dowworkflow , 25. UPsle sites , the conle sites  and 3D-Ele sites  to bring+ deviations. The brain regions implicated in each of the sensorimotor functions have been well characterized in previous work on N = 81 subjects at 3T, where robust and reliable activation across subjects was observed with a 5 min paradigm [The aim of the current study hence was to compare the performance of the SMS-EPI and the 3D-EPI sequences in detecting a range of task-evoked activations across different brain areas, with similar homogenized excitation profiles by using UPs. Metrics such as tSNR  and funcparadigm , with liparadigm , 34, andin-vivo exams with a sound pressure meter placed at a location equivalent to the right ear of the volunteers. In a pilot study, physiological data (cardiac pulsation and respiration) were recorded during the functional scans. As their inclusion in the analysis did not improve the detection of activation ROIs, they were not subsequently recorded.This study was approved by the local Institutional Review Board  and the volunteers provided informed written consent. Acquisitions were performed on 10 healthy volunteers  on a Magnetom 7T scanner  equipped with the Nova  8Tx-32Rx head coil. For each volunteer, a second order shim was performed, then an anatomical UP-enabled MPRAGE sequence  was acquwww.nmr.mgh.harvard.edu/software/c2p/sms), which uses sequential application of Slice-GRAPPA [z = 2), sagittal slice orientation, FA = 12° and no fat saturation owing to spatially non-selective water excitation [Imaging parameters of the UP-MPRAGE sequence were: TR/TE/TI 2600/3.4/1100 ms, flip angle (FA) 4°, 0.8 mm isotropic resolution, 192 sagittal slices, FOV 256 mm, parallel imaging with GRAPPA (R = 2), resulting in an acquisition time of 6:35 min. Besides a marginally longer TR, parameters of the SMS-EPI sequence closely matched the ones of the 7T Human Connectome Project (HCP) resting-state fMRI protocol [e-GRAPPA  and GRAPe-GRAPPA . The sam+ and ΔB0 field maps of 20 subjects that were collected in a previous study [Universal Pulses were designed for the SMS-EPI and the 3D-EPI sequences using the offline flip angle (FA) normalized root mean square error (NRMSE) minimization algorithm described in . The aveus study . The pulus study  in complus study .A waveform with two spokes for each slice was chosen for the design of the UP of the SMS-EPI sequence . The durThe functional localizer consisted of a fast event-related paradigm of 5:20 min duration as previously applied in the 3T study introduced above and used as reference . It was http://www.fil.ion.ucl.ac.uk/spm) [Data were preprocessed with SPM12   for all .uk/spm)  and tiss.uk/spm)  which ar.uk/spm) , 46. Thehttps://nilearn.github.io/) a CompCor [Univariate activation maps were obtained by fitting onto a general linear model (GLM). First, we only included the temporal onsets of each stimulus convolved with the canonical hemodynamic response function (default parameters in SPM) and their first derivative in order to account for latency in BOLD response. We calculated linear contrasts of parameter estimates to test the effect of each task (e.g. right vs. left button press) and tested their significance with mass-univariate (voxel-wise) t-tests. A p-value of 0.001, uncorrected for multiple comparisons, was chosen as the significance threshold since it is the threshold used for subject-level analysis in the study that we used as a reference . The mil CompCor  analysisThe SMS-EPI and 3D-EPI sequences were compared in terms of raw tSNR, t-score testing for the mean signal , activatActivation strength maps were computed by averaging the t-score maps for each contrast over the population. The contrasts were the following: video—audio , audio—video, left button press—right button press , right button press—left button press, video sentences—checkerboard  and computation—sentences .ref. The four sensorimotor contrasts that were shown to be robust versus inter-subject and inter-session variability were used for benchmarking. On the other hand, the higher level networks , though activated, were not used for this quantitative benchmark because of considerably higher intersubject variability [ref over the total number of voxels in ROIref. Specificity corresponds to 1—the rate of false positives; it was computed for each of the four sensorimotor contrasts as 1—the number of activated voxels outside ROIref over the total number of voxels outside ROIref. Note that both sensitivity and specificity depend on the significance threshold used to define activated vs. inactivated voxels. The sensitivities and specificities of the SMS-EPI and 3D-EPI sequences were computed for significance thresholds ranging from 0 to 0.5 to build the Receiver Operating Characteristic (ROC) curve [In this context, the SMS-EPI and 3D-EPI sequences were evaluated according to their capability to correctly detect activation, in comparison with the reference activation maps reported at 3T , for theiability . A good C) curve . Their rC) curve , the d’ in-vivo EPI raw images acquired over two subjects of the current study are provided in Normalized flip angle maps simulated with Bloch equations over the 20 subjects of the pulse design database are provided in The raw tSNR maps averaged over the subjects of the current study and normalized on the 2 mm isotropic MNI template are displayed in Brain areas activated by sensorimotor and higher level contrasts of the localizer and averaged over the subjects are displayed in SMS-EPI = 0.70±0.06 vs. AUC3D-EPI = 0.68±0.06 for the analysis performed with AR(1); AUCSMS-EPI = 0.70±0.05 vs. AUC3D-EPI = 0.70±0.06 for the analysis performed with FAST. The mean sensitivity, specificity and d’ results obtained with AR(1) and FAST noise whitening models for p<0.001 are reported in SMS-EPI = 1.23±0.29 vs. d’3D-EPI = 1.21±0.21 for AR(1) and d’SMS-EPI = 1.28±0.25 vs. d’3D-EPI = 1.28±0.19 for FAST. When the nuisance regressors were not included in the GLM analysis, these values read: d’SMS-EPI = 1.20±0.29 vs. d’3D-EPI = 1.12±0.23 for AR(1) and d’SMS-EPI = 1.29±0.15 vs. d’3D-EPI = 1.21±0.22 for FAST. The improvement of including the nuisance regressors in the GLM in terms of d’ was higher for the 3D-EPI (p = 0.06 and t = 2.17 for AR(1), p = 0.02 and t = 2.83 for FAST) compared to the SMS-EPI sequence (p = 0.47 and t = 0.76 for AR(1), p = 0.80 and t = 0.26 for FAST). For testing purposes, data processing was also performed with 0 and 2 mm smoothing kernels. Resulting d’ were lowered, especially for the 3D-EPI sequence: it was 36% and 11% lower without smoothing for the 3D-EPI and SMS-EPI sequences, respectively, while it was 8% and 1% lower with the 2 mm smoothing kernel for the 3D-EPI and SMS-EPI sequences, respectively. Reducing the smoothing kernel size particularly lowered the specificity of the 3D-EPI sequence.The ROC curves obtained by comparison of the activation maps to the reference ROIs and averaged over the four sensorimotor contrasts are displayed in In the current study, we showed that the performances of the SMS-EPI and 3D-EPI sequences to detect visual, auditory and motor contrasts were equivalent in a multi-task fMRI experiment performed at 7T and using pTx UPs, for matched spatial and temporal resolutions, highly similar to that of the resting-state fMRI protocol of HCP . In this+), pulse calculation (tailored pulse design) or other pTx-related steps (e.g. brain masking for pulse optimization). A study focusing on the improvements brought by UPs versus single channel quadrature excitations for the SMS-EPI and for the HCP-style resting-state fMRI protocol was also reported in [+-inhomogeneity mitigation schemes obviously depend on the sequence details. In this study, not employing such schemes would clearly have an impact on the comparison results.In the current study, the UPs allowed for straightforward and operator independent application to functional MRI, with neither time penalty for calibration scans  and not to the slice selection gradients. Other orientations for the SMS-EPI, e.g. as in , thus woThe signal variance of the fMRI time series originates from both thermal and physiological sources . Their dLimits of this study include the use of reference ROIs for the detection of functional activations: the ROIs obtained in  were conWe evaluated the SMS-EPI and 3D-EPI sequences with similar homogenized excitation profiles in a task fMRI study with matched spatial (1.6 mm) and temporal (1200 ms) resolutions at 7T. Complimentary to the other comparisons that can be found in the literature –13, the


Osteosarcoma is a common malignancy with high mortality and poor prognosis due to lack of predictive markers. Increasing evidence has demonstrated that pseudogenes, a type of non-coding gene, play an important role in tumorigenesis. The aim of this study was to identify a prognostic pseudogene signature of osteosarcoma by machine learning. A sample of 94 osteosarcoma patients’ RNA-Seq data with clinical follow-up information was involved in the study. The survival-related pseudogenes were screened and related signature model was constructed by cox-regression analysis . The predictive value of the signature was further validated in different subgroups. The putative biological functions were determined by co-expression analysis. In total, 125 survival-related pseudogenes were identified and a four-pseudogene ; RPL7AP28, HR: 0.32 (95% CI: 0.14–0.76); RP4-706A16.3, HR: 1.89 (95% CI: 1.35–2.65); RP11-326A19.5, HR: 0.52(95% CI: 0.37–0.74)) signature effectively distinguished the high- and low-risk patients, and predicted prognosis with high sensitivity and specificity (AUC: 0.878). Furthermore, the signature was applicable to patients of different genders, ages, and metastatic status. Co-expression analysis revealed the four pseudogenes are involved in regulating malignant phenotype, immune, and DNA/RNA editing. This four-pseudogene signature is not only a promising predictor of prognosis and survival, but also a potential marker for monitoring therapeutic schedule. Therefore, our findings may have potential clinical significance. Osteosarcoma is the most common malignancy derived from bone, which is originated in mesenchymal tissue like many other sarcomas. It usually occurs in children and young adults as well as the elderly with a typical bimodal distribution in age . The incPseudogenes are a class of homologues of the corresponding functional genes, which are also known as ‘gene fossils’ or ‘junk genes’. They belong to a subclass of long non-coding RNAs . Their iRecent development in bioinformatics and the availability of large-scale RNA-seq transcriptome data of multiple cancers with clinical follow-up data provide better approaches to explore the biomarkers for diagnosis and prognosis, allowing for a better understanding of the mechanism of cancer and improvement for patients’ outcome . HoweverIn our study, we applied machine learning analysis including univariate cox regression, LASSO cox regression, and multivariate cox regression analysis to construct a pseudogene-based signature to predict the prognosis outcome for osteosarcoma patients. First, by univariate regression, we identified survival related pseudogenes. Next, we narrow down the significantly prognosis related pseudogenes by LASSO regression and multivariate regression, from which we constructed a four-pseudogene based prognostic signature. Then we assessed the clinical utility of this prognostic model and explored its potential functions. Our findings provide new insights into predicting and evaluating the clinical outcome of osteosarcoma patients.https://ocg.cancer.gov/). In total, there are clinical information of 274 patients and RNA-seq data of 101 patients. TPM is used in this study which is a kind of RNA-seq data scaled by gene length and sequencing depth (M) [The osteosarcoma RNA sequencing (RNA-seq) expression data and the corresponding clinical follow-up data were obtained from the public database TARGET (epth (M) . After eepth (M) .p-value < 0.05 were screened as candidate pseudogenes for next analysis . These candidate pseudogenes were further screened by LASSO regression, which is an efficient method for regression analysis of high-dimensional predictors [p < 0.05 was selected for predictive signature construction. Corresponding risk score of each patient was calculated by the formula: risk score = expression of RPL11-551L14.1 × β (−0.4327) + expression of RPL7AP28 × β (−1.1344) + expression of RP4_706A16.3 × β(0.6360) + expression of RP11_326A19.5 × β (−0.6503). Patients were divided into high and low risk groups according to the median risk score (1.23). The Kaplan–Meier (K–M) survival analysis was performed on the two groups. Furthermore, the receiver operating characteristic (ROC) analysis was performed to evaluate the predicting efficiency of the model to predict 3-, 5-, and 8-year survival and the area under curve (AUC) was calculated. All of these processes were conducted by R software (version 3.5.1).Univariate cox regression analysis was performed to screen survival related pseudogenes. The pseudogenes with edictors , and wasedictors . 10-foldedictors ,30,31,32We combined several basic clinical information with the risk score signature to predict the 3-, 5-, and 8-year survival of osteosarcoma patients by plotting nomogram. Calibration plot, which demonstrates whether the predicted outcome is similar with the actual outcome, was used to evaluate the performance of the nomogram. In this section, R package rms was used to plot nomogram and calibration plot.https://david.ncifcrf.gov/summary.jsp) [p-value < 0.05 was regarded significant, which is described in detail in our previous studies [The Pearson correlation coefficients between the expression profiles of the four prognostic pseudogenes and protein-coding genes (PCGs) were calculated to determine the co-expression relationships of the pseudogenes and PCGs. The PCGs positively or negatively correlated with the four pseudogenes were considered as pseudogene-related PCGs for functional analysis. The online Database for Annotation, Visualization, and Integrated Discovery   was used studies ,21,34,35The osteosarcoma cohort includes data from a total of 274 samples. After excluding samples without clinical follow-up information, 94 osteosarcoma patients were involved in this study and the baseline of clinical characters were presented . The stup < 0.05 as the cut off, 125 pseudogenes were identified by the univariate analysis as significant to overall survival  were used to construct the model. A four-pseudogene  based risk model was finally obtained (N = 47 each) according to the median risk score  in both the male and female groups (N = 72) and older  patients  with the four-pseudogene signature for predicting patient survival, we constructed a multivariable cox probability hazard model to predict the 3-year, 5-year, and 8-year survival, and visualized it by a nomogram with an assigned score for each term A. Both tCorrelation analysis was used to identify the protein coding genes co-expressing with the pseudogenes, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to determine their function. The top 10 correlated PCGs of each pseudogene and their relationship are presented as a network A. The gep < 0.0001) ; MPV17L2, HR: 0.39 (95% CI: 0.24–0.65)) A,B. The  0.0001) C. ROC an 0.0001) D. Compar 0.0001) A. The RO 0.0001) B,C. Comp 0.0001) D.With the application of chemotherapy in the 1970s, the treatment of osteosarcoma has made great progress. However, survival rate of metastatic and relapse cases remains to be unsatisfactory, and the poor prognosis of such patients is the major problem for osteosarcoma . Thus, iThere are some limitations and shortcomings in this study that cannot be ignored. First, this study was mainly focused on data mining and data analysis, which are based on methodology and the results were not validated using experiments. Further experiments are needed to verify the findings of this study. Second, the datasets we were able to obtain were limited as we could only obtain one osteosarcoma dataset that contained both patient RNA-seq data and clinical follow-up information. If there were another dataset that matched our requirements, it could have been used to further validate our results. Additional datasets should be included to obtain a better result. Besides, there is currently no other study exploiting pseudogene signature for osteosarcoma, meaning that we also cannot validate our result in another independent study. Third, when constructing a pseudogene signature for prognosis, one must take it into consideration of the application of such a model. Since different methods of detecting pseudogenes might lead to different results, the procedure of detection, quantification, and determination of transcriptional activity of pseudogenes must be standardized . TherefoTaken together, we identified a novel four-pseudogene signature for osteosarcoma which is a promising independent survival predictor and served as an important biomarker for guiding the clinical treatment of osteosarcoma to improve management for patients. In addition, our findings provide new insights into exploring the underlying molecular mechanisms of osteosarcoma, and present a promising new prognostic marker. Therefore, our findings in the signature have a very promising clinical significance.


Considering the above, we studied the response of various strains of F. graminearum sensu stricto to different levels of l-pyroglutamic acid on solid YES (yeast extract sucrose) media. l-Pyroglutamic acid decreased the accumulation of trichothecenes in all examined strains. Gene expression studies addressing Tri genes , which induce the biosynthesis of trichothecenes, revealed the production of mycotoxins by l-pyroglutamic acid to be inhibited at the transcriptional level. Besides inhibitory effects on mycotoxin production, l-pyroglutamic acid exhibited variable and concentration-related effects on phenylpropanoid production by fungi. Accumulation of most of the fungal-derived phenolic acids decreased in the presence of 100 and 400 µg/g of l-pyroglutamic acid. However, a higher dose (800 µg/g) of l-pyroglutamic acid increased the accumulation of trans-cinnamic acid in the media. The accumulation of fungal-derived naringenin increased in the presence of l-pyroglutamic acid. Contrasting results were obtained for quercetin, apigenin, luteolin, and kaempferol, the accumulation of which decreased in the samples treated with 100 and 400 µg/g of l-pyroglutamic acid, whereas the highest l-pyroglutamic acid concentration (800 µg/g) seemed to induce their biosynthesis. The results obtained in this study provide new insights for breeders involved in studies on resistance against Fusaria.Fungicide application remains amongst the most widely used methods of fungal control in agroecosystems. However, the extensive use of fungicides poses hazards to human health and the natural environment and does not always ensure the effective decrease of mycotoxins in food and feed. Nowadays, the rising threat from mycotoxin contamination of staple foods has stimulated efforts in developing alternative strategies to control plant pathogenic fungi. A substantial effort is focused on the identification of plant-derived compounds inhibiting mycotoxin production by plant pathogenic fungi.  Fusarium graminearum sensu stricto (s.s.) remains a severe cereal pathogen worldwide. On small-grain cereals such as barley and wheat, this necrotroph can cause two different diseases: FHB  and FRR (Fusarium foot and root rot) [A common ascomycete fungus, oot rot) ,2. FHB coot rot) .Type B trichothecenes inhibit eukaryotic protein synthesis by binding to the 60S ribosomal unit and by interacting with the peptidyl transferase enzyme . ConsumpF. graminearum s.s. can develop increased resistance to this class of fungicides [The use of fungicides, principally azoles, is a major method for FHB management . The conngicides ,14,15,16ngicides .F. graminearum s.s. [In recent years, accelerated efforts have been undertaken into the development of environmentally friendly disease management approaches . Breedinrum s.s. ,29,30. l-pyroglutamic acid on the secondary metabolism of F. graminearum s.s. This organic acid was previously identified as a resistance-related (RR) metabolite in barley in response to Fusarium infection [l-Pyroglutamic acid is an analogue and a potential precursor of glutamate [l-pyroglutamic acid exhibits both antibacterial [Pseudoperonospora cubensis and Phytophthora infestans [In this study, we characterized the impact of nfection ,22,31. llutamate , which ilutamate . Previouacterial  and antinfestans . To the l-pyroglutamic acid on the accumulation of trichothecenes in the culture media as well as on the expression of the Tri genes  which induce their biosynthesis. We found that both gene expression and trichothecene accumulation were inhibited in all studied strains treated with l-pyroglutamic acid. In the present study, we also showed that the treatment with l-pyroglutamic acid impacts fungal phenolic acid and flavonoid profiles. The results obtained in this study provide new insights, especially for breeders involved in studies on resistance against Fusaria.We studied the effect of l-pyroglutamic acid reduced its concentration by 90–91%, 46–51%, and 56–60%, respectively. The substantial reduction of exogenous l-pyroglutamic acid might be linked to its efficient metabolization by fungi. l-pyroglutamic acid can be easily converted to glutamate by 5-oxoprolinase [After 21 days of incubation, fungi that were exposed to 100, 400, or 800 µg/g of rolinase ,37 and hrolinase . l-pyroglutamic acid, changes were observed in fungal secondary metabolite profiles. The total phenolic acids content decreased by 3.5- to 5-fold (depending on l-pyroglutamic acid concentration) in the fungal cultures exposed to l-pyroglutamic acid. It is worth noting, however, that in the case of two phenolic acids (protocatechuic and trans-cinnamic acids), exceptional results were obtained. No significant changes in protocatechuic acid were found between the samples treated with l-pyroglutamic acid and controls . The second exception concerned the higher concentration of trans-cinnamic acid after incubation with 800 µg/g of l-pyroglutamic acid. Nonoxidative deamination of l-phenylalanine to trans-cinnamic acid by phenylalanine ammonia-lyase  is a key step of phenylpropanoid biosynthesis. Accumulation of trans-cinnamic acid was also identified by Kulik et al. [Fusarium strains’ treatment with chlorogenic acid and by Bilska et al. [trans-cinnamic acid may indicate that the activity of PAL responsible for phenylpropanoid production was not completely inhibited by l-pyroglutamic acid. Increased accumulation of trans-cinnamic acid may also suggest that l-pyroglutamic acid has an inhibitory effect on enzymes converting this first intermediate of all phenylpropanoids further. Fungal strains used in this study produced phenolic acids and phenylpropanoids on the solid YES (yeast extract sucrose) medium , which ik et al.  after Fua et al.  after thl-pyroglutamic acid induced the biosynthesis of naringenin. Accumulation of other flavonoids  was inhibited in the samples treated with two lower concentrations of l-pyroglutamic acid (100 and 400 µg/g), whereas the highest acid concentration (800 µg/g) seemed to induce their biosynthesis. The sum of flavonoids increased almost 2.5-fold after incubation with 800 µg/g of l-pyroglutamic acid. It is unclear why the exposure to lower amounts of l-pyroglutamic acid suppressed the production of some flavonoids by fungal strains. However, the increased concentration of naringenin and other flavonoids may be indicative of trans-cinnamic acid conversion to naringenin chalcone, which is a precursor of naringenin  and chalcone isomerase (CHI)  , whereasl-pyroglutamic acid on the production of trichothecenes by fungal strains was established based on trichothecene counts determined on the treated and nontreated plates . l-Pyroglutamic acid had strong inhibitory effects on trichothecene accumulation in the case of all three studied F. graminearum s.s. strains; however, the degree of toxin inhibition was strain-dependent  expression was evaluated on day three of incubation  = 0.001) in most samples treated with 100 µg/g of l-pyroglutamic acid. Expression of Tri genes is regulated by two genes: Tri10 and Tri6 [Tri10 positively regulates the transcription of Tri6 [Tri genes [Tri10 transcription generally correlated with Tri4 and Tri5 expression; however, there were some exceptions. We noticed inhibition of Tri4 and Tri5 gene expression in MUCL 53455 strains in the presence of l-pyroglutamic acid, despite no changes observed in Tri10 expression. The effect of cubation . Gene exand Tri6 ,40,41. Tri genes ,40. In ol-Pyroglutamic acid decreased the accumulation of trichothecenes in all examined strains. Gene expression studies addressing Tri genes which induce the biosynthesis of trichothecenes  revealed the production of mycotoxins by l-pyroglutamic acid to be inhibited at the transcriptional level. Besides inhibitory effects on mycotoxin production, l-pyroglutamic acid exhibited variable and concentration-related effects on phenylpropanoid production by fungi.Fusarium graminearum s.s. strains used in this study are summarized in l-Pyroglutamic acid (dissolved in 10 mL of 96% ethanol)  was added to the YES (yeast extract sucrose) medium to obtain the final concentrations of: 100 μg/g, 400 μg/g, and 800 μg/g. The three acid concentrations corresponded to those used by Kulik et al. [l-pyroglutamic acid control , and three YES + fungal controls (F. graminearum s.s. strains incubated on YES media). k et al. ,29 and Bk et al. . Three dPetri dishes (Ø 80 mm) were inoculated from laboratory stock cultures (6–8 weeks old) and incubated at 25 °C (in triplicate) in the dark. Gene expression analyses were performed after 3 days of incubation, while chemical analyses  were performed after 21 days of incubation. l-pyroglutamic acid were determined using an internal standard at a wavelength of λ = 310 nm. l-Pyroglutamic acid identification was based on a comparison of sample and standard retention times. A specific amount of the standard was added to the analyzed samples. In the next step, the analysis was repeated. The detection level was 1 μg/g. The retention time of l-pyroglutamic acid was 19.05 min.Fungal-derived phenylpropanoid concentrations were established in dried fungal cultures after 21 days of incubation , as prevl-pyroglutamic acid were conducted according to the methods provided earlier by Kim et al. [VCEAC/L and ABTS+ assays of m et al.  and Re em et al. , respectl-pyroglutamic acid, as described in a study by Perkowski et al. [GC-MS was used to determine levels of trichothecenes  in fungal cultures exposed or nonexposed  to various concentrations of i et al. . l-pyroglutamic acid was used for RNA extraction. Six biological replications were prepared for each studied condition. Total RNA was extracted using a Pure Link® RNA MINI KIT  following the manufacturer’s recommendations. Extraction of RNA and reverse transcription were conducted according to Kulik et al. [Mycelium from 3-day-old fungal cultures grown on the YES medium exposed or nonexposed  to various concentrations of k et al. ,47. cDNATri4, Tri5) playing a fundamental role in trichothecene biosynthesis [Tri10 regulatory gene [Ef1α) was used as a reference gene. The experiment was conducted in accordance with Kulik et al. [Tri4, Tri5, and Tri10) genes in control and treated samples, relative to the Ct values obtained for the reference Ef1α gene. In this study, expression of two genes  was used to show the differences among mycotoxin levels. The production of phenylpropanoids (phenolic acids and flavonoids) was estimated by an analysis of the differences among phenylpropanoid levels between YES-only and YES + fungal controls using the Student’s t-test at p < 0.05. This statistical method was also used to test the impact of exogenously applied l-pyroglutamic acid on the accumulation of phenylpropanoids and to analyze the reduction of exogenous l-pyroglutamic acid. Statistical analyses were performed using STATISTICA . Tukey’s HSD (honest significant difference) test (


According to such procedure, adult male Sprague–Dawley rats were first exposed to one inhibitory avoidance apparatus and, with a 1-min delay, to a second apparatus where they received an inescapable footshock. Forty-eight hours later, retention latencies were tested, in a randomized order, in the two training apparatuses as well as in a novel contextually modified apparatus to assess both strength and generalization of memory. Our results indicated that both amphetamine and MDPV induced generalization of fear memory, whereas only amphetamine enhanced memory strength. Co-administration of the β-adrenoceptor antagonist propranolol prevented the effects of both amphetamine and MDPV on the strength and generalization of memory. The dopaminergic receptor blocker cis-flupenthixol selectively reversed the amphetamine effect on memory generalization. These findings indicate that amphetamine and MDPV induce generalization of fear memory through different modulations of noradrenergic and dopaminergic neurotransmission.Human studies have consistently shown that drugs of abuse affect memory function. The psychostimulants amphetamine and the “bath salt” 3,4-methylenedioxypyrovalerone (MDPV) increase brain monoamine levels through a similar, yet not identical, mechanism of action. Findings indicate that amphetamine enhances the consolidation of memory for emotional experiences, but still MDPV effects on memory function are underinvestigated. Here, we tested the effects induced by these two drugs on generalization of fear memory and their relative neurobiological underpinnings. To this aim, we used a modified version of the classical inhibitory avoidance task, termed  Indeed, both drugs of abuse have the same molecular targets represented by the norepinephrine (NE), dopamine (DA) and serotonin re-uptake transporters , but MDPV displays greater potency than amphetamine with regard to DA re-uptake transport .Male adult Sprague–Dawley rats  from Charles River Laboratories  were housed individually in a temperature-controlled (21 ± 1°C) vivarium room and maintained under a 12 h/12 h light/dark cycle (7:00 A.M. to 7:00 P.M. lights on). Food and water were available For all experiments, rats were trained and tested on a modified version of the classic inhibitory avoidance task, termed inhibitory avoidance discrimination task, that allows to investigate both strength and accuracy of memory  was recorded. No footshock was delivered in this box. Afterward, the rats were removed from the apparatus and, after a delay of 1-min, placed in the starting compartment of the second inhibitory avoidance apparatus (Shock box). We selected a 1-min delay because, as previously demonstrated -1-phenylpropan-2-amine; 1 and 3 mg/kg] and 3,4-methylenedioxypyrovalerone  were dissolved in saline (vehicle) and administered intraperitoneally, at the volume of 1 ml/kg, immediately after the training session . In the post hoc tests for between and within-group differences. P-values of less than 0.05 were considered statistically significant. The number of rats per group is indicated in the figure legends.Data are expressed as mean ± SEM. All data were analyzed with ANOVA for Repeated Measures (RM ANOVA) with drug treatment as between-group factor and retention latencies of individual animals in the different test contexts  as repeated measure. Two-way ANOVAs were used to analyze retention latencies of rats treated with propranolol vs. saline alone and cis-flupenthixol vs. saline alone. The source of the detected significances was determined by Tukey–Kramer F = 10.23, P < 0.01) as well as context  = 4.08, P = 0.02), but no significant interaction between these two factors  = 0.48, P = 0.75). Post hoc analysis, in accordance to what it has been previously demonstrated  and Non-Shock box (P < 0.01) compared to those in the Novel box, indicating that saline-treated rats were able to discriminate the two training contexts from the new one they had visited only during the test trial (P < 0.05), indicating that amphetamine, at the higher dose tested, enhanced the strength of memory. Furthermore, amphetamine (3 mg/kg)-treated rats showed longer retention latencies in both the Non-Shock box (P < 0.05) and Novel box (P < 0.01) compared to saline-treated animals. Thus, these results revealed that amphetamine-induced memory generalization across contexts.Rats were trained on the inhibitory avoidance discrimination task and given an immediate post-training intraperitoneal injection of amphetamine, MDPV or saline. With regard to amphetamine effects, as shown in st trial . RetentiF = 1.83, P = 0.18), a significant context effect  = 3.37, P = 0.04), and no significant interaction between these two factors  = 1.04, P = 0.39). Post hoc analysis confirmed that the performance of control animals was the same as for the amphetamine experiments (P < 0.05) in the Novel box. These results show that rats that were treated with MDPV (1 mg/kg) had similar retention latencies in all three boxes, indicating that MDPV induced generalization across contexts. Taken together, these findings indicate that amphetamine and MDPV have differential effects on memory strength, but that both drugs increase generalization of fear memory to a novel safe context.With regard to MDPV effects, as shown in eriments . RetentiAll training latencies are shown in We sought to test whether the amphetamine- and MDPV-mediated effects on strength and generalization of memory involved activation of the noradrenergic system. Herein, rats were given intraperitoneal injections of the β-adrenoceptor antagonist propranolol or saline 30 min prior to training, followed by post-training administrations of the effective doses of amphetamine (3 mg/kg), MDPV (1 mg/kg), or their corresponding vehicles.F = 4.80, P = 0.01). Similar to the control rats described above, post hoc analysis confirmed that saline-treated animals showed longer retention latencies in the Shock box (P < 0.05) and Non-Shock box (P < 0.05) as compared to those in the Novel box, thus indicating that control rats were able to discriminate the two training contexts from the new one that they visited only during the test trial. The same results were obtained with the RM ANOVA analysis for retention latencies of propranolol alone-treated animals  = 4.52, P = 0.02). Post hoc analysis revealed that propranolol alone-treated rats showed longer retention latencies in the Shock box (P < 0.05) and Non-Shock box (P < 0.05) as compared to those in the Novel box. These findings indicate that also rats that were treated with propranolol accurately remembered the two training contexts, even if they were not able to discriminate in which training context they received the footshock. Moreover, two-way ANOVA for retention latencies of rats treated with saline and propranolol did not reveal a significant treatment effect  = 0.59, P = 0.44) or treatment × context interaction effect  = 0.03, P = 0.97), but revealed a significant effect of the context  = 9.23, P < 0.0001), suggesting that treatment does not affect animals memory retention for different apparatuses  = 11.70, P < 0.01) as well as context  = 6.01, P < 0.01), and no significant differences for the interaction between both factors  = 0.50, P = 0.80). Retention latencies of rats treated with amphetamine alone in the Shock box (P < 0.05), Non-Shock box (P < 0.05) and Novel box (P < 0.01) were all significantly longer than those displayed by saline-treated animals in the same boxes. Retention latencies of rats that were treated with propranolol together with amphetamine in the Shock box (P < 0.05), Non-Shock box (P < 0.01) and Novel box (P < 0.01) were significantly shorter compared to those of animals treated with amphetamine alone in the same boxes. Moreover, retention latencies of rats treated with amphetamine alone in the Shock box (P < 0.05), Non-Shock box (P < 0.01) and Novel box (P < 0.01) were significantly longer than those of rats treated with propranolol alone in the same boxes.To investigate whether the noradrenergic system influences on amphetamine-mediated effects on memory generalization, we first analyzed retention latencies of saline- and propranolol alone-treated animals in the three contexts . RM ANOVaratuses . As showTo evaluate whether noradrenergic activity is also involved in the modulation of the MDPV effects on memory generalization, we analyzed retention latencies of both saline and propranolol alone-treated animals and confirmed the results that we described above for the experiments involving amphetamine . FurtherF = 1.70, P = 0.19) or treatment × context interaction effect  = 1.12, P = 0.36), but revealed a significant effect of the context  = 7.32, P < 0.01). Rats treated with MDPV alone showed longer retention latencies in the Novel box than those of saline alone- (P < 0.01) or propranolol alone-treated rats (P < 0.05) exposed to the same box. Moreover, retention latencies of animals treated with propranolol together with MDPV in the Shock-box were significantly longer compared to the Novel box (P < 0.05) and in the Non-Shock box compared to the Novel box (P < 0.05). Particularly in the Novel box, retention latencies of animals treated with propranolol together with MDPV were significantly shorter compared to those of MDPV alone-treated animals in the same box.As shown in In summary, these findings indicate that the amphetamine effect on enhancing memory strength is mediated by the noradrenergic system. Moreover, our findings indicate that the amphetamine effect on memory generalization appears to be only partially due to a modulation of the noradrenergic system, whereas the memory generalization effect induced by MDPV is entirely dependent on noradrenergic activity.All training latencies are indicated in In this set of experiments, we tested whether dopaminergic activity is involved in the effects induced by amphetamine and MDPV on memory generalization. To this aim, rats were intraperitoneally treated with the DA receptor antagonist cis-flupenthixol or saline 30 min before the training trial and subjected to post-training administration of the effective doses of amphetamine (3 mg/kg), MDPV (1 mg/kg), or their corresponding vehicle solutions.F = 10.87, P < 0.01) and context  = 17.62, P < 0.01), but not significant interaction between both factors  = 0.47, P = 0.83) effect. Post hoc analysis revealed that retention latencies of amphetamine alone-treated rats were significantly longer than those of rats that were given saline alone in the Shock box (P < 0.05), Non-Shock box (P < 0.05) and Novel box (P < 0.01). Retention latencies of rats that were treated with amphetamine alone were significantly longer than those of cis-flupenthixol alone-treated rats in the Shock box (P < 0.05), Non-Shock box (P < 0.01) and Novel box (P < 0.01). Retention latencies in the Novel box of rats treated with cis-flupenthixol together with amphetamine were significantly shorter with respect to rats given amphetamine alone (P < 0.01) in the same box. Moreover, they showed longer latencies in the Shock box and in the Non-Shock box compared to the Novel box (P < 0.05).As previously done in the experiments involving the noradrenergic system, we first analyzed the retention latencies of saline- and of cis-flupenthixol alone-treated animals in the three experimental contexts. Animals that were treated with saline showed comparable latencies to control groups that were discussed above . MoreoveF = 1.71, P = 0.18), a significant effect of the context  = 5.06, P < 0.01) and no significant interaction between these two factors  = 0.81, P = 0.56) effect. Post hoc analysis revealed that retention latencies of rats treated with MDPV alone were significantly longer than those of rats given saline alone and cis-flupenthixol alone in the Novel box (P < 0.01), and that the retention latencies of rats treated with cis-flupenthixol together with MDPV were significantly longer than those of rats given saline alone and cis-flupenthixol alone in the Novel box (P < 0.05).Concerning the dopaminergic role on MDPV-mediated generalization effects on memory, for the retention latencies of both saline- and cis-flupenthixol alone-treated rats, we confirmed the same results as described above ; again, In conclusion, these results demonstrated that the dopaminergic system is involved in modulating the effects of amphetamine on memory generalization as well with only a partial interference on its effects on memory strength. However, the blockade of DA receptors does not influence MDPV effects on memory generalization.All training latencies are shown in The present findings indicate that amphetamine and MDPV have different effects on memory strength, but both drugs increase generalization of fear memory to a novel safe context. We further show that noradrenergic and dopaminergic neurotransmission is differentially involved in the effects mediated by amphetamine and MDPV on memory. As previously showed, saline-treated animals trained in the inhibitory avoidance discrimination task, with a 1-min interval between the two training apparatuses, were able to discriminate the two training contexts from the new one visited only during the test trial (Atucha and Roozendaal, Previous studies have indicated that both amphetamine and MDPV, through a similar, yet not identical, mechanism of action increase brain monoamines release, particularly NE and DA, two neurotransmitters extensively involved in the modulation of memory (LaLumiere et al., Notwithstanding the different mechanism of action through which these two psychostimulants enhance NE and DA levels, both drugs of abuse enhance noradrenergic and dopaminergic neurotransmission (Robertson et al., via a different involvement of NE and DA neurotransmission. These results pave the way for future studies aimed at investigating the role of specific brain areas in mediating the differential effects of both psychostimulant drugs on strength and quality of memory, thus ultimately leading to reveal the neurobiological underpinnings of memory alterations induced by drugs of abuse.Brain regions with a high density of DAT and dopaminergic receptors, such as the striatum and nucleus accumbens (Efimova et al., The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Italian Ministry of Health.All authors contributed to and have approved the final manuscript. PCo and GM contributed to the design of the experiments, performed the experiments, analyzed data and wrote the manuscript. AS and CZ performed the experiments and analyzed data. AM and VT contributed to the design of the experiments. BR contributed to the design of the experiments and wrote the manuscript. PCa supervised the project, designed the experiments and wrote the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Enterovirus A species group. EV-A71 causes hand, foot and mouth disease (HFMD), with virulent variants exhibiting polio-like acute flaccid paralysis and other central nervous system manifestations. We analysed all enterovirus A71 complete genomes with collection dates from 2008 to mid-2018. All sub-genotypes exhibit a strong molecular clock with omega (dN/dS) suggesting strong purifying selection. In sub-genotypes B5 and C4, positive selection can be detected at two surface sites on the VP1 protein, also detected in positive selection studies performed prior to 2008. Toggling of a limited repertoire of amino acids at these positively selected residues over the last decade suggests that EV-A71 may be undergoing a sustained frequency-dependent selection process for immune evasion, raising issues for vaccine development. These same sites have also been previously implicated in virus-host binding and strain-associated severity of HFMD, suggesting that immune evasion may be an indirect driver for virulence (154 words).Enterovirus A71 (EV-A71) is an emerging pathogen in the  Enterovirus  contains many viruses pathogenic to humans. Historically, the most important have been the three human polioviruses, found within species Enterovirus C, but there are also several significant emerging pathogens1. Among these is Enterovirus A71 (EV-A71) which, along with around a dozen other viruses from species Enterovirus A and Enterovirus B2 causes hand, foot and mouth disease (HFMD). EV-A71 exhibits extensive genetic diversity, with genome sequences clustered into three established genotypes: A, B and C3. Genotypes B and C are also divisible into sub-genotypes: B1 to B5 and C1 to C5, respectively. A small number of outlier EV-A71 sequences have led to suggestions for the definition of additional genotypes and sub-genotypes5.The genus 6. The milder end of the clinical spectrum of EV-A71 infection in children presents as high fever and vomiting, together with the characteristic HFMD pattern of ulceration on the palms of the hands, soles of the feet and within the mouth. At the severe end of the range, brainstem and cerebellar encephalitis complications arise7, along with an acute flaccid paralysis that has led EV-A71 to be dubbed “the new polio”8. Further complications of cardiopulmonary failure lead to death in the most severe cases. With several large HFMD outbreaks in east Asian countries over the last three decades, totalling several million cases and thousands of fatalities9, EV-A71 constitutes an emerging pathogen with epidemiological impact far beyond that of classic milder HFMD caused by other viruses. Although vaccine development for EV-A71 has been a research priority for nearly two decades11, there is no universally licensed vaccine or antiviral treatment for EV-A71 infection12.HFMD surveillance in China from 2008–2014 showed that EV-A71 was the major aetiological agent of the disease, in at least 44% of cases19. Searches for positive selection have also been carried out23 and there is some overlap between the detected residues in the two types of study, suggesting that the virulence of EV-A71 may be undergoing natural selection.A further research priority in recent years has been elucidation of the molecular basis of neurovirulence in EV-A71. Such knowledge would enable prediction of the likely severity of new outbreaks, or prognosis in individual cases where the viral genome sequence could be determined24. Supportive of this notion, epidemics of EV-A71 often correlate with the appearance of a previously unencountered sub-genotype in a population - for instance the successive epidemics of sub-genotypes B3, B4 and B5 in Singapore25 and the cyclical nature of outbreaks of EV-A7126. Tee et al.20 show that EV-A71 phylogenetic trees display a ladder-like morphology, suggesting that immune evasion may be a selective pressure producing antigenic drift within EV-A71 cyclical sub-genotypes.Evolution of EV-A71 at the molecular level has implications for vaccine design. EV-A71 sub-genotypes are serologically distinctive, implying that acquired immunity to one sub-genotype may not be completely protective in future outbreaks of different sub-genotypes27, suggesting that human populations may differ in susceptibility. This may help to explain why separation of virulent and non-virulent strains purely on the basis of viral genome sequence, has remained elusive.The preponderance of large epidemics of virulent EV-A71 in east Asia, despite the presence of the virus on all continents, is unexplained. Host factors such as HLA-A33 and HLA-DR17 responses have been linked to severe HFMDet al.20, which covered all EV-A71 sequences up to mid-2008, by analysing EV-A71 genome sequences from 2008 to mid-2018. We examine the behaviour of the molecular clock within sub-genotypes, and use Bayesian skyline analysis to demonstrate population dynamics of the two most important sub-genotypes of the last decade. We then search for evidence of amino acid residues under positive selection, and display their patterns of variation on phylogenetic trees. The structural consequences of positive selection on EV-A71 proteins are examined and implications for immune evasion, antigenic drift and virulence are discussed.In this report, we update the analyses of Tee 28, averages at 0.30 to 0.33 substitutions per site, between EV-A71 genotypes. Within genotypes, the corresponding figure is 0.17 to 0.19 substitutions per site between sub-genotypes. The genetic distance from EV-A71 to CV-A16, EV-A71’s nearest relative within species Enterovirus A, ranges from 0.35 to 0.43 substitutions per site, and to baboon enterovirus A13, the outlier in Enterovirus A, from 0.85 to 0.91 substitutions per site  dates of 2001 for B5 and 1992 for C4. The Bayesian skylines suggest that B5 expanded around 2007 and then again from 2012 to 2014. C4, by contrast shows a steadier expansion from the early 1990s, peaking around 2004 with a decline in the early part of the present decade, coincident with the second expansion of B5.Figure Table Within this general pattern of selective constraint, positive selection is detected on certain sites. Figure 30 at its vertex. Between the “mountain” and another raised feature is a “canyon”. An antibody binding site is found on both slopes of the canyon31.Figure Figure Our sampling protocol for EV-A71 sequences used in this analysis, is directed at all complete genomes over the last decade, where collection dates are available. This reduces the 12122 total sequences from EV-A71 in GenBank on our sequence retrieval date to a subset of 697, from which a further 46 suspected recombinant genomes were removed. The stringency of this protocol is designed to ensure data quality, but carries the risk of sampling bias. We note that Table 32, as previously suggested by Tee et al.20. We calculate that overall rates of omega (dN/dS) in EV-A71 sub-genotypes have been low since 2008 . 697 qualifying sequences were found to have collection dates. Rapid input of sequences sets to TempEst for molecular clock analysis is assisted by the following expedient. Where only year of collection is given, TeL generated a collection date of 30th June for that year . Where only a month and year of collection is given, TeL generated the 15th of that month .Sequences with a collection date were then extracted using E-type (available in the Supplementary Data), which compares each sequence with a subset of the reference genomes given by van der Sanden, et al.36 plus MG67247837 for putative genotype E, using needle from EMBOSS38 to perform a global pairwise alignment. The needle output is parsed for each reference genome and the closest match was used to assign a sub-genotype to the query sequence. As output, E-type produced a separate FASTA-formatted file for each sub-genotype. Where the difference between the closest sub-genotype match and the second closest match for any sequence was < = 5% (152 sequences), further analysis was carried out using Simplot39. If Simplot indicated a likely recombination event, the sequence was removed from the analysis (46 sequences).TeL-dated output sequences were grouped into sub-genotypes using duptrimmer (available in the Supplementary Data). This is a rare occurrence, found once in genotype B5 and twice in C4.Duplicate sequences are also removed from each sub-genotype file if they share the same collection date, using E-type with ten or more qualifying sequences after the removal of duplicates and recombinants, were aligned using MAFFT40, and a maximum likelihood tree produced in MEGA41 using the GTR + G substitution model42. TempEst32 was used to derive the best fitting root and assess clock-like behaviour for each sub-genotype tree. For those sub-genotypes with more than 25 qualifying sequences (only C4 and B5), Bayesian skyline plots were produced using BEAST43 and Tracer44.Sub-genotypes output files from 45 (version 1.5.0). An initial estimate of the transition/transversion bias (kappa) was obtained in MEGA and was used along with an initial value of omega (dN/dS) of 1  in the Slr input parameters. After the first run, the values of kappa and omega derived by Slr were used as input parameters to confirm convergence. Visualization of positively detected sites was performed in Molecular Operating Environment (MOE), 2013.08 .Genome sequences with a collection date before 2008 were removed and each sub-genotype alignment was trimmed to the boundaries of the coding sequence. Where necessary, alignment was refined using Muscle in “align codons” mode within MEGA. Positive selection, neutrality and constraint were analysed on a site-wise basis using Slr